Mint plant &#39;Indus&#39;

ABSTRACT

The present invention relates to a new and distinct mint plant of  Mentha piperita  ‘Cim Indus’, selected through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm, possessing the characters of producing high amount of menthofuran ranging between 22 to 30% of total oil content, high amount pulegone ranging between 9.0 to 18% of total oil content, with essential oil content ranging between 0.32 to 0.40% of the total oil content.

FIELD OF THE INVENTION Genus, Species, and Variety

The present invention relates to a new and distinct mint plant of thegenus Mentha, species piperita, designated the variety ‘Cim Indus’.

This variety was selected through screening, field trial and analysis ofmonoterpene constituents of the essential oil of the germplasm. Thisvariety possesses the characters of producing high amount of menthofuranranging between 22 to 30% of total oil content, high amount pulegoneranging between 9.0 to 18% of total oil content, with essential oilcontent ranging between 0.32 to 0.40% of the total oil content.

BACKGROUND OF THE INVENTION

Menthofuran (3,6-dimethyl-4,5,6,7-tetrahydrocoumarone) is one of themajor constituent for aroma of the essential oil extracted from theleaves of Mentha piperita. Because any other compound has not duplicatedthe aroma effect, menthofuran is important in the formulation of certainstandardized essential oils, such as peppermint oil. However,menthofuran is an expensive compound of limited availability as theplants produce 0 to 6% menthofuran (U.S. Plant Pat. No. 11,788).Literatures are available for the chemical synthesis of menthofuran tosubstitute the naturally available menthofuran (U.S. Pat. No. 4,240,969) to reduce cost of production. Also the acceptability of syntheticmenthofuran is a limiting factor in determining the cost of theessential oil mixture containing synthetic components in aroma industry.

Considering the importance of menthofuran in aroma industry under ‘NewMillennium Indian Technology Initiative (NMITLI) programme’ launched byCouncil of Scientific and Industrial Research (CSIR), India, during2001, a systematic approach was undertaken to evaluate the existinggermplasm of M. piperita at CIMAP and breed for genetic enhancementtowards high menthofuran biosynthesis in the essential oil. Systematicbreeding experiments to allow open pollination followed by single seedprogeny selection by chemotypic evaluation for enhanced constituent(menthofuran) led to development of this chemotype the variety ‘CimIndus’.

OBJECTS OF THE INVENTION

The main object of the present invention is to develop a new anddistinct mint plant.

Another main object of the present invention is to develop a novel mintplant through screening, field trial and analysis of monoterpeneconstituents of the essential oil of the germplasm.

Yet another object of the present invention is to develop a plantproducing high amount of menthofuran.

Still another object of the present invention is to develop a plantproducing high amount pulegone.

Still another object of the present invention is to develop a plantproducing high herbage.

Still another object of the present invention is to develop a mint plantshowing resistance against major plant disease conditions like leafspot, rust, powdery mildew, lepidopteran pest Spilarctia obliqua.

SUMMARY OF THE INVENTION

The present invention relates to a new and distinct mint plant of Menthapiperita ‘Cim Indus’, selected through screening, field trial andanalysis of monoterpene constituents of the essential oil of thegermplasm, possessing the characters of producing high amount ofmenthofuran ranging between 22 to 30% of total oil content, high amountpulegone ranging between 9.0 to 18% of total oil content, with essentialoil content ranging between 0.32 to 0.40% of the total oil content,

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows cluster analysis for the chemotype “Cim Indus” compared to‘Kukrail’, Tushar’, and ‘Pranjal’.

FIG. 2 shows the RAPD profile of the Mentha piperita genotypeCIMPA/MP20.

FIG. 3 shows a twig of CIMAP/MP20

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a new and distinct mintplant of Mentha piperita ‘Cim Indus’, selected through screening, fieldtrial and analysis of monoterpene constituents of the essential oil ofthe germplasm, possessing the characters of producing high amount ofmenthofuran ranging between 22 to 30% of total oil content, high amountpulegone ranging between 9.0 to 18% of total oil content, with essentialoil content ranging between 0.32 to 0.40% of the total oil content,

The plant was developed at the CIMAP research farm, Lucknow, India andasexually propagated through suckers for further planting in the samefarm. By appearance, this new variety is a typical plant of Menthapiperita. The difference between this new variety and the parent orother known varieties is in the herbage at menthofucan, pulegone andother essential components. Thus, this is a new and distinct mint plantof Mentha piperita variety ‘Cim Indus’, selected through screening,field trial and analysis of monoterpene constituents of the essentialoil of the germplasm, possessing the following combination ofcharacters:

-   a. the said plant produces high amount of menthofuran ranging    between 22 to 30% of total oil content,-   b. the said plant produces high amount pulegone ranging between 9.0    to 18% of total oil content,-   c. the said plant produces essential oil content ranging between    0.32 to 0.40% of the total oil content,-   d. the said plant produces herbage yield ranging between 200-220    quintal per hectare,-   e. the said plant is of height ranging between 65 to 70 cms, with    plant canopy of area ranging between 78-85 cms,-   f. the said plant shows resistance against leaf spot, rust, powdery    mildew, lepidopteran pest Spilarctia oblique,-   g. the said plant has Quadrangular, woody stems, of color purplish    green with RHS color code of 59A, the surface texture of the stem is    hairy and rough,-   h. the said plant has simple, opposite, and decussate leaves, of    with an upper surface of dark green color with color code of RHS    137A, and a lower surface of green color with color code of RHS    137C; the surface of the leaf is rough and hairy with purple violet    colour (81 A) on lower veins;-   i. the said plant has leaves of chartaceous texture,-   j. the said plant's leaves has Glabrous dorsal surface, with hairy    on ventral veins,-   k. the said plant has leaves of ovate-elliptical shape, with serrate    margins,-   l. the said plant has leaves with acute-acuminate tip, obtuse base,    and broad size,-   m. the said plant has leaf with petiole of length ranging between    0.5 to 0.9 cm,-   n. the said plant has leaf with of area about 7.91 cm²,-   o. the said plant has leaf with length ranging between 1.2 to 4.3    cm,-   p. the said plant has leaf width ranging between 0.6 to 2.6 cms,-   q. the said plant has inflorescence of nature terminal spike,-   r. the said plant has flowers of following traits:    -   i. arranged in whorls,    -   ii. smooth pedicel,    -   iii. green color pedicel with RHS code 137B,    -   iv. calyx is glabrous, tubular, 5-lobed, margin ciliated, yellow        green with RHS color code of 146C,    -   v. corolla is tubular, 4-lobed, with subsequeal lobes, and is        light purple tending towards white, whitish purple: 76 D;    -   vi. flowers are whitish purple: 76 D,    -   vii. anthers are four in number, exserted, grayed-red with RHS        code of 181A,    -   viii. stigma is bifid.-   s. the said plant is able to produce higher herbage, menthofuran and    pulegone yield per unit area as compared to other existing improved    varieties,-   t. the said plant produce high menthofuran when harvested 75 days    after planting and 115 days after planting,-   u. the said plant produce high pulegone when harvested 75 days after    planting,-   v. the said plant is able to produce higher pulegone and menthofuran    due to up regulation and thus has the potential to isolate    regulatory factors for monoterpene metabolism, and-   w. the said plant has distinct molecular profile by random amplified    polymorphic DNA (RAPD) using 20 random primers (OPA) distinguishing    the plant from the other existing varieties.

The flowers are arranged in whorls and the inflorescence grow which varyin length. The flowers include:

-   -   -   Pedicel.—1.0 to 1.5 mm in length, Yellow green (145C);        -   Calyx.—Four sepals, persistent, 1.0 to 2.5 mm in length,            Yellow green (145B).        -   Calyx diameter.—1 mm.        -   Calyx texture.—Rough.        -   Corrolla.—Whitish purple (76D) 2.0 to 4.0 mm in length,            composed of 4 petals, differentiated into tube and a limb.        -   Corolla texture.—Smooth.

-   Androecium:    -   -   Anthers.—Four, ocidimetary, white, remain inside the corolla            tube.

-   Gynoecium:    -   -   Stigma.—Bifid, bicarpellary syncarpous.        -   Ovary.—superior, deeply four-lobed, bilocular.        -   Style.—gynobasic arising between the lobes of the ovary.        -   Stigma.—Red-Purple Group 71C.        -   Color of ovaries.—Yellow-Green Group 151A.

The leaves have predominantly the carvone and menthofuran smell. Thisvariety produces no fruit and no seeds.

The present invention is related to the development of a novel highmenthofuran and pulegone producing chemotype which can also yield highamount of pulegone through proper harvest management. The plantchemotype was obtained through screening of the open pollinated seedprogenies of the variety ‘Kukrail’. The invention is further related tothe plant producing more herb yield leading to higher production ofessential oil per unit area compared to the seed parent variety. Theselected plant possesses the property of accumulating more menthofuranand pulegone at specific developmental stages and hence propermanagement prior to processing can yield high amount of these importantphytochemicals for industrial use. This plant is unique and clearlydistinct from all other existing varieties of Mentha piperita. The newplant type ‘Cim Indus’ can be propagated vegetatively through suckersand runners for commercial cultivation.

‘Kukrail’ is a released variety of CIMAP which is maintained along withthe germplasm of CIMAP in the field systematically every year. Everyyear in the month of October, the twigs are planted in small sized plots(3 m×3 m) for generation of enough planting material for planting in themain field during the month of January. Open pollinated seeds arecollected from different genotypes every year and analyzed formonoterpene constituents in the essential oil. CIMAP/MP20 is such agenotype selected from open pollinated seed progenies of the variety‘Kukrail’. Mentha piperita is propagated vegetatively through runners.With the NMITLI initiative the runners generated from the seed plotswere planted in 5 m×5 m plots during the month of January 2001,following normal agronomic practices with the objective to screengenotypes rich in menthofuran in the essential oil. Replicated samplesfrom each genotypes were taken from the field by planting multipliedrunners in the month of January, 2001, 2002 and 2003 for 3 consecutiveyears in RBD fashion and different growth and yield characteristics wererecorded (Table 1). For field trials the replicated plots were preparedby adding only FYM 1.5 ton per ha. The three-year averages of herbyield, essential oil yield and the variations in major essential oilcomponents are detailed below for the genotype CIMAP/MP20 compared tothe CIMAP released varieties of Mentha piperita ‘Kukrail’, ‘Tushar’,‘Pranjal’. ‘Pranjal’ bears the U.S. Plant Pat. No. 14,090. TABLE 1Comparative herbage, oil, menthofuran and pulegone yield of Menthapiperita genotypes. Genotypes CIMAP/MP20 Kukrail Tushar Pranjal Oilcontent (%) 0.35 0.40 0.63 0.55 Herrbage yield (Quintal/ 206.8 123.8190.5 123.8 hectare) Oil yield (Litre per 72.41 49.52 119.04 68.10hectare) Menthofuran content(%) 27.24 8.727 9.648 8.385 CalculatedMenthofuran 19.72 4.32 11.484 5.710 yield (Litre per hectare)Pulegone(%) 15.405 3.032 2.355 2.783 Calculated Pulegone 11.155 1.5012.80 1.89 yield (Litre per hectare)

If menthofuran is aimed the genotype can yield highest amount of naturalmenthofuran than any other variety released and reported which is thecase for pulegone also.

Oil Extraction and GLC Analysis

Oil samples from the field grown plants were extracted byhydrodistillation using Clevenger's apparatus and weighed to record theyield. Over ground shoot samples were collected from the whole plantselected randomly from the middle of the row of each replicated plots atdifferent days after planting (35. 55, 75, 95, 115 days after planting).Shoots collected from individual genotypes were bulked for eachtreatment plot and essential oil was distilled from all the replicatestaking 500 g of bulked shoots containing leaves. The final analysis ofall the essential oil samples was accomplished on Varian CX-3400 using a30 m×0.25 mm Supelcowax-10 column. The injector and detector temperaturewere maintained at 200 and 225° C. respectively, with oven temperatureprogrammed from 60 to 200° C. at the rate of 7° C. min⁻¹ increase, withinitial and final holds of 2 and 5 minutes respectively. Hydrogen gaswas used as carrier at the rate of 1 ml min⁻¹ and an aliquot of thesample was injected with a split ratio of 1:50. Data were processed inthe electronic integrator Varian 4400 and the identification was basedon retention time of authentic samples of 1-menthol (Takasago, Japan)and retention indices calculations (Jennings W & Shibamoto T (1980)Qualitative analysis of flavour and fragrance volatile by capillary GC,Academic Press Inc., New York.). TABLE 2 Variation in major essentialoil components of Mentha piperita genotype CIMAP/MP20 at differentstages of growth. 95 35 DAYS 55 DAYS 75 DAYS DAYS 115 DAYS Limonene0.6405 6.5948 5.6849 5.6283 4.484 Menthone 1.4922 48.55 2.0802 3.82981.9770 Menthofuan 1.4922 6.2713 23.96 3.4181 27.246 Menthol 32.568134.138 12.7920 28.2289 14.396 Pulegone 9.6714 0.2421 10.4367 16.883615.405

TABLE 3 Comparative monoterpene component profiles in the essential oilof M. piperata P(20), Kukrail, Tushar, Pranjal, 115 days after planting.Components CIMAP/MP20 Kukrail Tushar Pranjal 1 α-Pinene 0.432 0.4650.661 0.707 2 β-Pinene 1.004 0.874 1.256 1.472 3 Sabinene 0.857 1.0310.786 0.881 4 Myrcene 4.897 0.342 0.342 0.341 5 α-terpinene 0.081 0.2440.089 0.069 6 Limonene 4.484 2.862 2.656 3.145 7 1,8 Cincole 8.809 4.9045.112 5.252 8 γ- 0.769 0.226 0.244 0.248 Terpinene 9 p-Cymene 0.3040.309 0.105 0.130 10 3-Octanol 0.133 0.127 0.258 0.322 11 Menthone 1.97021.292 28.248 28.339 12 Menthofuran 27.246 8.727 9.648 8.385 13 Iso0.556 3.963 4.410 4.084 menthone 14 Menthyl 2.323 7.857 4.768 3.799acetate 15 Neo menthol 4.757 3.870 2.897 3.288 16 Caryophyllene 0.6610.452 0.088 0.056 17 Pulegone 15.405 3.032 2.355 2.783 18 Menthol 14.39628.840 26.818 26.147 19 Piperitone 1.536 2.331 1.056 1.232 20 Carvone0.606 0.376 0.585 0.796

The genotype CIMAP/MP20 has a characteristic oil profile which expressesdifferentially at different stages of growth. The menthofuran contentwas found to be higher at 75 days stage which decreased during 95 daysand again increased during harvesting time 115 days. Correspondingmenthol content in the essential oil content was found to be negativelycorrelated to the menthofuran content at corresponding stages of growth.

Pulegone content increased after 75 days and was stabilized after 95 to115 days (Table 2). The comparative monoterpene profiles for differentcomponents is presented in Table 3 during harvesting time (115 daysstage).

Trichhome Analysis of the Genotype CIMAP/MP20

Monoterpenes are known to be cytotoxic to plant tissues, inhibitingrespiration and photosynthesis by drastically affecting themitochondria, golgi bodies etc and decreasing cell membrane permeability(Brown J T, Hegarty P K & Charlwood B V (1987) The toxicity ofmonoterpenes to plant cell cultures. Plant Science 48:195-201.).Monoterpenes are either sequestered in the plants in specializedstructures like glandular hairs in Pelargonium (Brown J T & Charlwood BV (1986) Differentiation and monoterpene biosynthesis in plant cellcultures. In; Morris P, Scragg A, Stafford A and Fowler M (eds)Secondary Metabolism in Plant Cell Cultures. Cambridge University Press,Cambridge, 1986, p. 68.), trichomes in Mentha or stored in the form ofnon-toxic glycoside derivatives in vacuoles e.g. Rosa spp.

So the number of trichomes at different developmental stages of thegenotype CIMAP/MP20 and its variation in different leaves (both theupper and lower surface) situated at different level (0 level: leaf atthe tip, 1 level: next leaf down to 0 level, 2 level: next leaf down to1 level, 3 level: next leaf down to 2 level, 4 level: next leaf down to3 level,) were characterized and finally all the trichome at differentlevels of the leaves were averaged, calculated per centimeter squareleaf area. A peak trichome density was observed from 75 to 95 days inall the leaves at different levels except the leaf at the tip. At 0level the leaves are at active developmental stage which may be causefor steady rate for trichome formation (Table 4). TABLE 4 Trichomedensity(Trichomes/cm²) in the leaves of the genotype CIMAP/MP20 atdifferent developmental stages. 35 days 55 days 75 days 95 days 115 daysLevels stage stage stage stage stage 0 1416 2816 5499 5392 5400 1 12232592 4373 5112 3106 2 982 1349 2443 2464 2392 3 875 1168 1813 2080 16684 610 752 1824 1205 1477

Taxonomic description of the peppermint plant CIMAP/MP20 are as givenbelow:

-   1. Genus: Mentha.-   2. Species: piperita.-   3. Family: Lamiaceae.-   4. Common name: Peppermint.-   5. Plant height: 65-70 cm.-   6. Plant canopy: 80-84 cm.-   7. Growth habit: Herbaceous, erect and branched.-   8. Stem: Quadrangular, woody, purplish green (59A).-   9. Leaf: Simple, opposite, decussate.    -   -   Colour.—Dark green (137A).        -   Texture.—Chartaceous.        -   Surface.—Glabrous dorsal surface, hairy on ventral veins.        -   Shape.—ovate-elliptical.        -   Margin.—serrate.        -   Tip.—acute-acuminate.        -   Base.—Obtuse.        -   Size.—broad.        -   Petiole length.—0.5 cm-0.9 cm.        -   Area.—7.91 cm².        -   Length.—1.2 cm-4.3 cm.        -   Width.—0.6 cm-2.6 cm.-   10. Leaf: stem ratio: 1.06.-   11. Inflorescence: Terminal spike.-   12.Flowers: Arranged in whorls.    -   -   Pedicel.—smooth, green (137B).        -   Calyx.—Glabrous, tubular, 5-lobed, margin ciliated,            yellowgreen(146C).        -   Corolla.—purple, tubular,4-lobed,lobes subequeal, white.        -   Anthers.—four, exserted, Grayed-red (181A).        -   Stigma.—Bifid.

The colour codes are in accordance with the “RHS colour chart publishedby The Royal Horticultural Society, 80 Vincent Square, London SW1P2PE,1995. The genotype CIMAP/MP20 was named and referred as ‘Cim Indus’in this specification.

DNA Isolation and PCR Amplification Reactions

DNA was isolated from leaf tissue essentially according to the protocoldescribed previously (Khanuja S P S, Shasany A K, Darokar M P & SushilKumar (1999) Rapid Isolation of PCR Amplifiable DNA from the Dry andFresh Samples of Plants Producing Large Amounts of Secondary Metabolitesand Essential oils by Modified CTAB Procedure. Plant Molecular BiologyReporter 17: 74.) and pooled DNA (equal amount from 20 individual plantsof a genotype in a field) constituted the samples for polymerase chainreactions (PCRs) which were carried out in 25 μl volume.

A reaction tube contained 25 ng of DNA, 0.2 unit of Taq DNA polymerase,100 μM each of dNTPs, 1.5 mM MgCl₂ and 5 p mol of decanucleotideprimers. The amplifications were carried out using the DNA Enginethermal cycler (MJ Research, USA) following the protocol of Khanuja etal. (Khanuja S P S, Shasany A K, Srivastava A & Sushil Kumar (2000).Assessment of genetic relationships in Mentha species. Euphytica 111:121-125.). The amplified products were loaded in 1.2% agarose gelcontaining 0.5 μg ml⁻¹ of ethidium bromide and photographed by Polaroidsystem. Twenty decamer primers procured from Operon Technologies, USA(OPA) were used to detect polymorphism in the selected genotype. Thesimilarity matrix obtained after multivariant analysis using Nei andLi's coefficient (Nei, N. & W. Li, 1979.

Mathematical model for studying genetic variation in terms ofrestriction endonucleases. Proc. Natl. Acad. Sci. (USA) 76: 5269-5273.)is shown in Table 5. These similarity coefficients were used to generatea tree for cluster analysis using UPGMA method (FIG. 1) which shows thedistinctiveness of the genotype CIMAP/MP20. The program ‘NTSYS 2.1’ wasemployed to generate the cluster diagram from the similarity indices.The genotype CIMAP/MP20 was 54.1%, 50.2% and 51.1% different with thevarieties ‘Kukrail’, ‘Tushar’ and ‘Pranjal’ respectively establishingthe uniqueness of the genotype. These primers were also used to developa unique RAPD profile of the chemotype ‘Cim Indus’ (FIG. 2). The bandsimilarities were not derived from a photograph but were calculateddirectly from the RAPD profiles from the agarose gels based on which thecluster diagram was made. TABLE 5 Similarity between the genotypescompared through RAPD profile analysis. Kukrail Tushar PranjalCIMAP/MP20 Kukrail 1.000 Tushar 0.960 1.000 Pranjal 0.950 0.960 1.000CIMAP/MP20 0.459 0498 0.489 1.000

FIG. 3 illustrates the RAPD profile of the Mentha piperita genotypeCIMPA/MP20. In this Figure Lane 1 represents the λ Hind III marker andlanes 2 to 21 represent, respectively, the profile for each of OPA 01through OPA 20. The identities of the primers employed in OPA 01 throughOPA 20 are provided in the accompanying sequence listing as SEQ ID NO:1through SEQ ID NO:20, respectively.

Uniformity and Stability

Like any other Mentha piperita genotype this genotype is also plantedvegetatively through runners and suckers. No variation of any kind wasobserved in this genotype for the last 3 years of trial maintaining thequality of oil and phenotype. The RAPD analysis of random plant samplesin different years of trial also did not show any variation in profilesfor this genotype indicating the stability of this genotype.

Disease and Pest Resistance

The incidence of lepidopteran pest Spilarctia obliqua and fungus mintrust (Puccinia sps), leaf spot and mildew were not detected in the fieldcontinuously for 3 years in the genotype CIMAP/MP20.

Metabolic Regulation of Menthofuran Biosynthesis

The genotype CIMAP/MP20 was rich in pulegone and menthofuran. In thebiosynthetic pathway Geranyl pyrophosphate is converted to limonenewhich in turn is transformed into isopiperitenol, followed by pulegone.Pulegone is converted to menthone followed by menthol. Menthol andMenthone are the main constituents of the essential oil of Menthapiperita. In one branch of the pathway pulegone is converted tomenthofuran. In this genotype the monoterpene pulegone is biosynthesizedat an accelerated rate and the reaction favour more towards thementhofuran synthesis than menthol. In the initial stage (up to 55 daysstage) of growth of this genotype the reaction favours for theproduction of menthol at a reduced rate from pulegone with lessaccumulation of pulegone and menthfuran. But at later stage as the plantmatures the reaction favours accumulation of more menthofuran andpulegone and instead the biosynthesis of menthol decreases. Thisindicate the role of regulatory proteins in the monoterpene metabolismand the importance of this genotype for the isolation of such proteinfor future modification of metabolic pathway modification.

1. A new and distinct mint plant of Mentha piperita variety ‘Cim Indus’,substantially as illustrated and described herein.